PIWI proteins and their associated small RNAs (piRNAs) are essential for male fertility in mammals. My long term goal is to understand the biology of piRNAs and their function in male fertility. Unknown mechanisms determine that some RNAs are loaded into PIWI and become piRNAs, while others are not. In this grant, I propose to study the structural and mechanistic factors that determine piRNA formation. I hypothesize that both (1) the unique structure of piRNA-producing loci and (2) specific post-transcriptional processing events target a transcript for piRNA production. Yet, neither is well understood. In the K99 phase of this proposal, I will study piRNA promoters, piRNA precursors, and piRNA processing intermediates. For these studies, I will develop new strategies for deep sequencing and fluorescent in-situ hybridization. I also propose to set up a cell-free system that recapitulates piRNA production for biochemical studies. In preliminary studies, I have already defined the transcription unit of piRNA producing loci, allowing their comparison with other genes, and I have observed a fragmentation event probably responsible for 5' end formation of piRNAs, perhaps coupling with PIWI loading. In the R00 phase of this proposal, I will identify the elements and events that are necessary and sufficient for piRNA production, and dissect the steps and locations of piRNAs metabolism in the context of spermatogenesis. The proposed study to define a piRNA will help us to understand why piRNA mutant mice are infertile, leading to a better understanding of human male infertility.